MODIFICATION OF COMPOSTING PROCESS OF CHICKEN MANURE USING NATURAL AND MG-MODIFIED ZEOLITE TO GENERATE A VALUE-ADDED PRODUCT WITH SUFFICIENT NUTRIENT CONTENT by Kehinde Simisola Idim B.Sc., Covenant University, 2010 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE IN ENVIRONMENTAL SCIENCE UNIVERSITY OF NORTHERN BRITISH COLUMBIA August 2021 © Kehinde Simisola Idim, 2021 Abstract This research was aimed at developing a composting process of organic waste using natural and modified zeolites by ascertaining how effective they were in retaining phosphorus (P) after 60 days. The experiments included compost (C) containing natural (N) and modified (M) zeolite (Z) treatments (applied at 10% and 15% on a weight basis of the total waste): CNZ10, CNZ15, CMZ10, CMZ15 and C (which is the control treatment with no zeolite addition). The second objective was to compare the barley shoot biomass and nutrient concentration of the various compost treatments mixed with soil and control treatment (just soil) after 45 days of planting in the greenhouse. In general, the benefits of co-addition of compost and zeolite on sandy soil were tested in a completely randomized experimental design. The results indicated that the treatments with the highest zeolite treatments (CNZ15 and CMZ15) proved most effective at retaining P by the end of the composting process (2.8mg and 2.9mg respectively) compared to the lower zeolite ratios and the control treatment; CNZ10 (2.0mg), CMZ10 (1.9mg) and C (0.7mg). The barley shoot biomass results indicated that the treatments that had a combination of soil (S) and modified zeolite (SCMZ10 and SCMZ15) had biomass of 7.67g and 7.24g respectively, followed by the natural zeolite and compost treatments (SCNZ10, SCNZ15 and SC) having 6.19g, 6.38g and 5.99g respectively and were significantly different from the control treatment (S) which had the lowest biomass of 0.48g. With respect to the plant nutrient concentration, N and P were significantly higher in the control treatment (S) compared to the other treatments while K concentration was significantly highest in the compost and some of the zeolite treatments (SC, SCNZ15 and SCMZ15) compared to other treatments. These results were in line with some previous findings where zeolite was used as a soil amendment in agriculture. Keywords: Composting, Natural Zeolite, Chicken Manure, Barley, Nutrient Concentration i Table of Contents Abstract ....................................................................................................................................... i Table of Figures ....................................................................................................................... iii List of Tables ............................................................................................................................ iv Acknowledgements .................................................................................................................... v 1. Introduction ............................................................................................................................ 1 1.1 Composting ...................................................................................................................... 1 1.2 Chicken manure................................................................................................................ 3 1.3 Natural zeolites ................................................................................................................. 3 1.3.1 Zeolite in Agriculture ................................................................................................ 6 1.4 Research Objectives: ........................................................................................................ 7 2. Materials and Methods:.......................................................................................................... 8 2.1 Zeolite Modification ......................................................................................................... 8 2.2 Zeolite Characterization ................................................................................................... 8 2.2.1 Elemental analysis by acid digestion ......................................................................... 9 2.3 Composting materials ....................................................................................................... 9 2.3.1 Composting process ................................................................................................... 9 2.4 Analytical techniques ..................................................................................................... 11 2.5 Soil sampling and characterization ................................................................................ 14 2.5.1 Physical Analysis ..................................................................................................... 14 2.6 Greenhouse Pot Experiment ........................................................................................... 16 2.6.1 Plant Growth Data ................................................................................................... 17 2.6.2 Shoot and Root Nutrient Content Analyses ............................................................. 17 2.7 Statistical Analyses ........................................................................................................ 18 3. Results and Discussions ....................................................................................................... 19 3.1 Elemental Composition of Natural Zeolite .................................................................... 19 3.1.1 XRD Analysis .......................................................................................................... 19 3.1.2 XRF analysis............................................................................................................ 19 3.2 Change in Temperature .................................................................................................. 21 3.3 pH Variation ................................................................................................................... 22 3.4 Electrical Conductivity (EC) .......................................................................................... 23 3.5 Final total carbon (C), total nitrogen (N) and C/N ratio for all treatments .................... 25 3.6 The effect of natural and modified zeolite on Nitrate Concentration ............................ 28 ii 3.7 The effect of natural and modified zeolite on NH4-N .................................................... 29 3.8 Effect of natural and modified zeolite on mehlich-3 extractable final P concentration of the compost after 60 days: .................................................................................................... 31 3.9 Shoot Growth ................................................................................................................. 32 3.9.1 Shoot Nutrient Analysis........................................................................................... 35 3.10 Root Growth: ................................................................................................................ 44 3.10.1 Root Nutrient Analysis: ......................................................................................... 45 4. Conclusion and Recommendation ....................................................................................... 52 Biblography.............................................................................................................................. 53 Appendix .................................................................................................................................. 64 Table of Figures Figure 1. View showing all fifteen reactors inside the growing chamber ............................... 10 Figure 2. Samples in the oven set at 105ºC for 24h ................................................................. 12 Figure 3. Soil saturation: Buchner funnel was used to extract soil water after 24hr ............... 14 Figure 4. XRD analysis for NZ (Natural Zeolite) and MZ (Modified Zeolite) ....................... 19 Figure 5. Variation in temperature during the first ten days of composting ............................ 21 Figure 6. Changes in pH during the composting process ........................................................ 23 Figure 7. Changes in EC during the composting process ........................................................ 24 Figure 8. Final C, total N and C/N Ratio ................................................................................. 27 Figure 9. NO3-N concentration after composting .................................................................... 29 Figure 10. NH4 -N Concentration. ......................................................................................... 30 Figure 11. Concentration of mehlich-3 Phosphorus (P) in the final compost after 60 days of composting. .............................................................................................................................. 32 Figure 12. The shoot height and shoot biomass for different treatments. ................................ 33 Figure 13. Pictorial representation of all the six different treatments after 45 days of planting .................................................................................................................................................. 35 Figure 14. Plant concentration of nutrients among the treatments .......................................... 41 Figure 15. The root biomass for different treatments: ............................................................. 44 Figure 16. Root concentration of nutrients among the treatments. The root total carbon and total nitrogen for different treatments ...................................................................................... 49 iii List of Tables Table 1. Physiochemical properties of chicken manure .......................................................... 10 Table 2. Compost treatments ................................................................................................... 11 Table 3. Physiochemical properties of the soil ........................................................................ 15 Table 4. Planting soil treatments .............................................................................................. 17 Table 5. The XRF analysis of NZ (natural zeolite) and MZ (modified zeolite) ...................... 20 Table 6. Properties of compost produced after 60 days ........................................................... 28 Table 7. Mehlich-3 concentration of final compost ................................................................. 31 iv Acknowledgements It was indeed an interesting experience these past few years of my research journey at the University of Northern British Columbia (UNBC). I am first grateful to God Almighty who was and will always be my guide. Thank you to the UNBC office of research for the RPA which helped cover the cost for some aspect of my research. I am incredibly grateful to my supervisor Dr Hossein Kazemian who encouraged & supported me and never gave up on me. To my co-supervisor Dr Ron Thring, thank you for your encouragement and support, very much appreciated. Thank you to my committee member Dr Mike Rutherford for your support and assistance through this journey even when it was under such short notice. Sincerely grateful to Doug Thompson and John Orlowsky, you are both amazing humans because your selflessness and willingness to make things easy always overwhelmed me. Thankful to my NALS colleagues: Erwin Rehl & Charles Bradshaw for their assistance and coordination during laboratory analysis. Thank you Dr Chris Opio for that reference letter and guidance which contributed to my success in my RPA application. I wish to express my profound gratitude to the management of Pacific Regeneration Technologies (PRT), Red Rock, B.C, Canada for allowing me into their nursery field to collect soil samples which was used for my planting process. Thank you to Dwayne Anchikoski for providing me with some chicken manure from his farm located at 9710 Johnson Road, Prince George who I got connected to through my colleague: Lon Kerr, you are both sincerely appreciated. I thank my friends/colleagues: Mostafa Marzi, Jingjing Shi and Farzana Nargis for their contribution in my research, you are all very much appreciated, and I could not have done this without your inputs. I thank my dad: Kola Olabowale for his financial support and words of v encouragement, I am indeed grateful Sir. To my kids: Isaac & Elizabeth, I dedicate this to you to let you know you can achieve whatever you set your mind to and thanks for loving me unconditionally also for the times Isaac asked if he could help me with my schoolwork, I love you both so much and I am grateful for the gift of you. Thank you to my husband for your support and helping with the kids when you could, I appreciate you. Thanks to my mum: Abiola Olabowale and my siblings: Taiwo Olabowale and Oreoluwa Afe for their support and constant words of encouragement. To all my other friends that supported and contributed to this research, I am grateful. vi 1. Introduction 1.1 Composting A world bank report about waste generation (projection for 2025) portrayed that a continuous increase in waste production in all regions is expected (Hoornweg & Bhada-Tata, 2012). Increasing global production of waste is as a result of urbanization, population and the economic growth (Zorpas et al., 2017). Studies carried out at waste management sites and landfills indicate that over 50% of wastes produced by households and small scale industries are organic wastes that can mostly be recycled and reused as compost (Chatterjee et al., 2013). Composting which is a biological oxidative process is generally accepted and carried out as a fast, simple and safe solution to transform agricultural, industrial and municipal organic wastes, to end products that contribute to soil conditioning and richness (Gamze Turan & Nuri Ergun, 2007). Composting has been established as one of the most widely used, eco-friendly and economical practical processes for adequate management of organic solid waste (OSW) that can stabilize organic matter whose nutrient can be used as organic fertilizers and/ or soil conditioners (Soudejani et al., 2019). . It involves the aerobic degradation of complex organic matter (OM) into simpler ones which eventually yield mature organic compost because of different microbes such as bacteria and fungi (Sadef et al., 2016). The microbial degradation being monitored is a very important step towards achieving the successful operation of the composting process (Wei et al., 2014). According to previous studies, parameters such as temperature, pH, moisture content, nutrient content and C/N ratio have proven to be very instrumental in the determination of the microbial development and organic matter degradation (M. P. Bernal et al., 2009). The application of compost in the agricultural sector can add to sustainable soil health (Kamyab et al., 2015). Composting decreases waste, eliminates weed 1 seeds, provides adequate sanitation and yields valuable end products of great importance in agriculture (Hubbe et al., 2010; Jiang et al., 2011; Sun et al., 2014). The emissions of greenhouse gases such as methane (CH4) have however been linked to the composting of organic wastes as a result of the degradation of soluble lipids, carbohydrates, organic acids and proteins in anaerobic conditions (Jia et al., 2016);(Fukumoto et al., 2011). The rapid degradation of available OM at the beginning of the composting process contributes to the chain of events that causes intensive acidification and slowing down of the composting process (Waqas et al., 2019). However, several research studies have proven that the addition of various additives and bulking agents to deal with these limitations and modify the physical structure of the composting matrix (Q. Wang et al., 2016). Compounds such as lignite (Chen et al., 2015), alum (Lefcourt & Meisinger, 2001a), carbon-base products (glucose, sucrose and straw powder) (Y. Li et al., 2013), medical stone (Q. Wang, Awasthi, Zhao, et al., 2017), glucose, sucrose, starch (Meng et al., 2016), biochar (Awasthi, Wang, Chen, et al., 2017; Awasthi, Wang, Pandey, et al., 2017), clay (Witter & Lopez-Real, 1988a), lime with struvite salts (X. Wang et al., 2018), magnesium and phosphate (Lee et al., 2009), phosphogypsum (Lim et al., 2017), bentonite (R. Li et al., 2012), hydrothermally treated lignocellulose (Nakhshiniev et al., 2014), earthworms(J. Wang et al., 2014) and natural zeolite (Awasthi, Wang, Huang, et al., 2016; Awasthi, Wang, Ren, et al., 2016; M. Bernal et al., 1993; Gholamhoseini et al., 2013a; Lefcourt & Meisinger, 2001b; Stylianou et al., 2008; Turan & Ergun, 2008; M. Wang et al., 2017; Q. Wang, Awasthi, Ren, et al., 2017; Q. Wang et al., 2018; Witter & Lopez-Real, 1988b) are studied as potential additives that can enhance the compost quality (Soudejani et al., 2019). Co-composting poultry manure with zeolite would be a potential solution to the challenge of global warming. Organic materials such as leaves, biochar, wood barks as well as inorganic materials like zeolites and other minerals are mostly used as bulking agents during the composting process in order to optimize and produce an adequate biological, chemical and 2 physical properties to the composting matrix (Dias et al., 2010). The use of natural zeolite due to its unique physicochemical characteristics is in recent times getting significant attention compared to other bulking agents applied to the composting matrix (Awasthi, Pandey, et al., 2016). 1.2 Chicken manure Chicken manure is the feces of chicken (sometimes mixed with bedding materials) which is known to have high concentration of nitrogen (N), phosphorus (P) and potassium (K). It is mostly essential for land application as an organic fertilizer as a result of its somewhat high nutrient content (Griffiths, 1998). It can be used as a fertilizer but mostly after it has been composted because if directly applied to the soil, it affects the root and other parts of the plant. It has been shown that aerobic composting is a successful method used to transform livestock manure into excellent organic fertilizer which can help to attain the goals of reduced, safe and useful utilization of manure. Manure poses a bigger threat because of ammonia emission when compared to food waste during composting because of its higher nitrogen content. The main issue is how to make full use of the benefits of chicken waste as an organic fertilizer and in the process reduce the risk it poses to the environment (Hochmuth et al., 2009). 1.3 Natural zeolites Zeolites are naturally occurring hydrated aluminosilicate minerals that possess a porous structure having important physicochemical properties such as sorption, cation exchange capacity, bulking agent and molecular sieving (Chan et al., 2016; Nizami et al., 2016). The structure of zeolites consists of three-dimensional frameworks of SiO4 and AlO4 tetrahedra (Erdem et al., 2004). The first zeolite was identified in 1756 by the Swedish mineralogist (Baron) Friedrich Axel Cronstedt, who noticed that on heating the stones he had put together, in a blow-pipe flame, they danced about in a froth of hot liquid and steam, appearing like the 3 stones themselves were boiling (Rhodes, 2007). In recent years, zeolites have received much attention in the scientific community because of their special physicochemical properties, available source and low cost, and their applications in industry, agriculture and pollution control (Wen et al., 2016). Zeolites contain alkali and alkaline-earth elements which exist in over 50 and 150 natural and synthetic forms respectively (Jha & Singh, 2016). The application of zeolites in the soil (both individually and in combination with minerals and organic fertilizers) not only increases the capacity of the crops but also improves their qualitative indices (Andronikashvili et al., 2010). Zeolites are good additives to the soil which helps to keep adequate level of nutrients in the soil and prevents it from leaching into water resources. Substitution of silicon by aluminum atoms in the crystal framework leads to extra negative charge to be balanced by surrounding counterions (such as Na+, K+, Ca2+, and Mg2+), and these counterions are easily exchanged by other surrounding cations in a contact solution (Tsitsishvili, 1992). The presence of competing cations, like Na+, K+, Ca2+ and Mg2+, adversely affects ammonium adsorption capacity of zeolites (Huang et al., 2010). Unlike other soil amendments, zeolite does not break down over time rather it remains in the soil to improve nutrient retention (Polat et al., 2004). Natural zeolites generally have a lower adsorption capacity, hence there is usually need for them to be modified before use to improve their adsorption capacity and purity (Huang et al., 2014). According to research, various modification processes were used to improve natural zeolite’s sorption capacity for many applications (Soudejani et al., 2018). Studies have shown that zeolite has high cation-exchange capacity (CEC) and high void volume which remains constant during wetting and drying cycles. Reports have in recent time shown that clinoptilolite (a type of zeolite) addition increased phosphorus availability to plants as a result of combined effect of fertilizer dissolution and ion exchange processes (Lancellotti et al., 2014). Increased phosphate 4 concentrations have been linked to increased rates of plant growth. Phosphorus is one of the necessary nutritional elements during plant growth (Schindler, 1974). Some studies such as Kuroda (2004) and Yasuda (2009), have made attempts to decrease the NH3 emissions that occur as a result of the composting process because NH3 is the primary contributor of odor in the composting process (Hanajima et al., 2010). Struvite crystallization (MgNH4PO4.6H2O) is initiated due to the addition of magnesium (Mg) and phosphate (PO 4) during composting and its effect on reducing NH3 emission have been proven in the composting of food waste, poultry, and swine manure (Jeong & Kim, 2001; W. Zhang & Lau, 2007). Contamination of nitrate in both underground and surface waters is because of nitrogen loss from irrigated cropland especially the sandy soils. Phosphorous is an essential element and often limiting nutrient in crop production, so it is one of the key fertilizer constituents (B. Li et al., 2019a). Poultry manure contains larger amounts of phosphate and magnesium than food waste and these are helpful for struvite crystallization. Struvite is a white crystal containing magnesium, ammonium, and phosphorus in equal molar concentrations (molecular weight = 247.42g/mol). Different physicochemical parameters can impact the formation of the struvite crystals such as the pH of the solution, mixing energy, temperature as well as the presence of foreign ions (Le Corre et al., 2009). Struvite can be described using the general reaction below: + + +6 → .6 (Scavia et al., 2014). The foremost advantage of struvite crystallization is the potential of promoting the recovered product which can be used as a fertilizer, material for building or adsorbent. The most important minerals in struvite are nitrogen (N) and phosphorus. Struvite as a fertilizer may be used to control release of nutrient, minimize loss of nutrient, and therefore supports the plant growth in a sustained way (B. Li et al., 2019b). 5 Not all soil types are adequate for agricultural practices hence modifying the soil physical properties can enhance nutrient movement in especially light-textured soils (Mamedov et al., 2016; Q. Wang et al., 2016). Nutrients should be retained in the soil to provide adequate nutrients for plant growth and leaching of these nutrients should be avoided (Bakhshayesh et al., 2014; Leggo, 2015; Nakhli et al., 2014). Leached mineral fertilizers (nutrients) are likely to cause water pollution problems (Kaleeem Abbasi et al., 2015). 1.3.1 Zeolite in Agriculture Crop yield increase and decrease in nitrate pollution of water systems are easily achievable through zeolite addition as a soil amendment (fertilizer) which is cost-effective for future improvement (Jakkula & Wani, 2018). Natural zeolite was first reportedly used for plant growth in Japan (Minato, 1968a). Soil macronutrients such as nitrogen, phosphorus and potassium can be dispensed to plants in the form of ammonium (NH4+) and Phosphate (P) exchanged zeolites (E. Allen et al., 1995, 1996; E. R. Allen et al., 1995). Zeolite’s most important application in agriculture is its ability to act as a slow-release fertilizer. Slow release has been described as delayed-release, controlled-release, controlled-variability, slow acting and metered-release (Ming & Allen, 1999). Zeolite when applied in collaboration with conventional fertilizers produced an increase in seed germination and an all-round increase in spinach yield (Spinaciaoleracea) (Burriesci, Valente, Ottana, et al., 1984). It was observed in a study conducted in southern Italy that zeolite had an outstanding effect which produced an increase in fruit size and yield of plums (Prunuspersica) and vines (Vitisvinfera) upon its application with traditional fertilizers (Burriesci, Valente, Zipelli, et al., 1984).As a result of zeolite’s unique features, it has been proposed that zeolites would be an ideal substrate for nutrient sorption and improvement of the soil moisture conditions (Ramesh & Reddy, 2011). Zeolites have high void volume that does not change during wetting and drying cycles(Essington, 2015). These voids in the zeolite form long and wide tunnels which are 6 similar to honeycomb that help to improve the movement of ions to and from the mineral structure (Polat et al., 2004). Differential addition experiment of zeolites in alfalfa fields showed that the 20% zeolite ratio to the 80% soil ratio generated the highest yield, taller crop and more developed root system(Turk et al., 2006).Literature survey strongly suggests that the co-addition of compost and zeolites depict a good practice of sustainable agriculture by increasing the nutrient availability and improving soil water retention capacity (Litaor et al., 2017). 1.4 Research Objectives: For the present study, we used barley (Hordeum vulgare L.). Co-amending soil with manure and zeolite can be a profitable method towards reducing chemical fertilizer applications and improving the sustainability of agricultural systems (Gholamhoseini et al., 2013). The objectives of this thesis research were to prove (i) how effective natural and modified zeolites are in retaining available Phosphorus (P) in the compost (final concentration) in comparison to the control which had no zeolite additions, (ii) the significant difference between the barley plant (Hordeum vulgare) biomass of compost and zeolite treatements in comparison to the control treatment and (iii) the significant difference in macronutrients and micronutrients concentrations between the various treatments. 7 2. Materials and Methods: 2.1 Zeolite Modification The natural zeolite used was obtained from Bromley deposit in British Columbia, Canada. It was passed through a 2mm sieve. According to the supplier’s technical datasheet, the chemical composition of the zeolite: SiO2=66.70, Al2O3=11.21, BaO=4.34, K2O=3.72, Fe2O3=1.76, CaO=1.65, Na2O=1.16, MgO=0.49, SiO2/Al2O3=5.95, and loss on ignition (L.O.I) =8.22 which confirms that it mainly consists of clinoptilolite. Natural zeolite was modified using (MgSO4.6H2O) salt (Huang et al., 2014). Prior to the modification process, the zeolite sample was washed with DI water to remove impurities such as ash and sand specimen, then oven dried overnight at 105°C. Subsequently, 2kg of dried natural zeolite was suspended in 20L of 0.1M (MgSO4.6H2O) solution. The suspension was stirred continuously over a period of 24 hours. The solution was decanted and thoroughly washed with DI water until the water was clear. The modified sample was then dried in the oven at 105 C ̊ for 24 hours. The modified zeolite was also characterized using XRD technique. 2.2 Zeolite Characterization Both natural and modified zeolite samples were crushed into fine powders to identify the physicochemical properties of the zeolite samples used in this study. X-ray Diffraction (XRD) was performed using Rigaku Miniflex 300 to identify the crystalline phase of zeolite samples. The quantitative chemical analysis was determined using the X-ray Fluorescence (XRF) to ascertain the chemical composition of the natural and modified zeolite samples. Both XRD and XRF were carried out in the Northern Analytical Lab Services (NALS) facility. 8 2.2.1 Elemental analysis by acid digestion To determine the elemental composition of the natural and modified zeolites, they were digested using reverse aqua regia (RAR) method. This acid regime consists of nitric acid (HNO3) to hydrochloric acid (HCl) molar ratio of 3:1. Concentrations of acids were 67– 70 %(w/w) for HNO3 and 34 –37 %(w/w) for HCl. The samples were crushed into fine powders and transferred into a 15ml plastic vial and the acid solutions were added to achieve the acid regime of RAR. Acid digestion process was carried out using digestion blocks (Digi prep) for 6 hours via automated control of temperatures which gradually heat up the sample vials as high as 95°C. After all the vials cooled down to room temperature, DI water was used to top up to the 15 mL mark on each vial to prevent further gassing of acids. All vials were shaken manually and then centrifuged at 600 rpm for 30 min to separate the nondigested zeolite constituents mainly silica. The supernatants were then collected and analyzed by ICP-OES (the results are presented in the appendix section). 2.3 Composting materials Chicken manure (green waste) was collected from a local farm located in Prince George, British Columbia, Canada. Sawdust combined with simple dried leaves (brown waste) were collected on UNBC campus. The chopped dried waste leaves were added as a source of bulking agent (An et al., 2012). The physiochemical characteristics of the chicken manure are presented in table 1. The compost mix was manually agitated twice every week for the duration of the process. 2.3.1 Composting process Chicken manure, saw dust and chopped simple dried waste leaves were mixed at a ratio of 1:0.5:0.05 (wet weight basis) respectively. Two different ratios (10% and 15%) of both natural and modified zeolites were added into the above-mentioned feedstock. The treatments without zeolite (control) were identified as C, the treatment with natural zeolite at mass ratios 9 of 10% and 15% were identified as CNZ, the treatment with modified zeolite at mass ratios of 10% and 15% were identified as CMZ. The treatments are summarized in table 2 below. Each of the five treatments had three replicates summing up to a total of fifteen reactors. Composting was conducted in separate but identical reactors for a period of 60 days. The reactors had a height of 14.5 cm, an inner diameter of 13.6 cm and an effective volume of 1.2 L. They were made from plastic and each reactor was covered with aluminum foil at the top as removable lids with small holes uniformly distributed (0.2 in diameter). This was to help prevent them from drying up and to allow proper inflow and outflow of oxygen. For every reactor, holes were drilled at the bottom and placed into another reactor to allow for possible leachate throughout the composting process. Table 1. Physiochemical properties of chicken manure Parameters Value Moisture Content (MC) 45% pH 6.5 Electrical Conductivity (EC) 1.3 dS/m Carbon 40% Nitrogen 2.1% Figure 1. View showing all fifteen reactors inside the growing chamber 10 The experiment was conducted at the Enhanced Forestry Laboratory (EFL) situated within UNBC campus. The reactors were placed inside a growing chamber under controlled temperature of 35± 2 C ̊ (Wei et al., 2014) and a relative humidity of 60%. Temperature was measured using a digital thermometer by inserting the thermometer in the middle of the pile within the reactor. The treatments are shown in table 2. Table 2. Compost treatments Treatments C CNZ10 CNZ15 CMZ10 CMZ15 Ratio of zeolite (% wt) in composting materials 0 10 15 10 15 C: Compost, NZ: Natural Zeolite, MZ: Modified Zeolite Approximately 2g of samples were collected on days 0, 14, 28, 44 and 60 from each reactor. The samples were taken from three different positions and homogenized manually to obtain a representative sample. The mixed sample was subdivided into two parts. One part was used to determine the immediate analysis such as pH, EC, OM while the second part was freeze-dried, grounded, and sieved for further analysis. After maturing, the composts were passed through a 2mm standard sieve prior to being used for soil amendment studies. The speed of composting and qualities of the finished compost depends largely on the selection and mixing of the raw materials. The mass of the compost differed because of the different densities of the feedstock (comprising of the chicken manure, saw dust, dried leaves, natural and modified zeolites) based on the various treatments. 2.4 Analytical techniques Temperature which is responsible for the vigorous microbial activity was measured every day throughout the composting process. A digital thermometer was used to carry out this process. For the evaluation of the compost maturity, some grams of the fresh compost sample were 11 taken from each reactor throughout the experimental period. The collected samples were subjected to different analyses such as: pH, electrical conductivity (EC), organic matter (OM), total carbon (TC), total nitrogen (TN), ammonium (NH4 –N), nitrate (NO3 –N). Moisture content (MC) was determined by weighing an empty aluminum plate, weighing out some grams (g) of samples into the aluminum plate. This was placed in an oven set to a temperature of 105 ºC for 24 h. The dried samples were then taken out of the oven and weighed after the oven was turned off. The MC which is the net percent loss in fresh weight was calculated by subtracting the final weight from the initial weight and dividing by the initial weight multiplied by 100% (Chan et al., 2016). Figure 2. Samples in the oven set at 105ºC for 24h The dried samples after MC determination were burnt for 3hr at 550 ºC in a muffle furnace to determine the OM. The percent loss in weight of the samples (volatile components) was calculated as OM. The total organic carbon and nitrogen contents were analyzed (Costech 4010 CHNSO analyzer) after drying the samples at 55 C ̊ for 72hr in an oven (Leco Corporation, 2006). The analyzer was used to combust the samples via the standard sequential combustion/ reduction setup recommended by Costech company with a flowrate of 100ml/min of helium 5.0 as the carrier gas. 12 To determine the pH and EC, some grams of the compost samples (1:10 w/v sample – deionized water ratio) were shaken for 1hr using a rotational shaker (Mupambwa & Mnkeni, 2015). The pH and EC of the aqueous extracts of the fresh compost samples were measured. The pH was determined using the pH probe of a benchtop Thermo Scientific Orion Star A211 pH meter while the EC was measured using a conductivity meter (Myron L 512M5). Ammonium nitrogen (NH4 -N) was extracted with 2M KCl solution (1:10 w/v sample-extractant ratio) and measured using auto analyzer (Bran Luebbe Auto Analyzer 3) (McKeague, 1978). Reverse aqua regia method (USEPA, 1996) was used for the determination of metal composition. Compost samples were crushed into a fine texture and dried at 55 C ̊ for 72hr. 0.2g of sample was weighed and transferred to a 15mL digestion tube. Trace grade strong mineral acids consisting of nitric acid (HNO3) and hydrochloric acid (HCl) were used for digestion at molar ratio of 3:1respectively. Every 10 samples contained method blank, spike, duplicate and sample spike. The samples were block digested (Digi prep) for 6 hours via automated control of temperatures, which gradually heats up the sample vials up to 95 ºC. When the vials cooled down to room temperature, DI water (type 1 water: 18.2MΩ) was used to top up to the mark of 15mL mark to prevent further gassing of acids. The vials were shaken manually and centrifuged at 600rpm for 30 min. The concentration of elements in the solutions were analyzed by running on the inductively coupled plasma optical emission spectrometer (Agilent Technologies 5100 ICPOES). Mehlich-3 extraction procedure was used to estimate the concentration of available nutrients to the plants. This was done by weighing 2g from each reactor into a 50mL Centrifuge tube then adding 20ml of Mehlich-3 extracting solution which was thoroughly mixed using a shaker. The solution was filtered, and the supernatant was stored in the fridge for ICP-OES analysis (Mehlich, 1984). Samples were weighed out and added to DI water which was equivalent to 13 10 times the weight of the compost samples into 15mL tubes. The mixture was stirred using a shaker and then pre-filtered through 0.45µm syringe filter and run on the Ion Chromatography (IC) DIONEX ICS 5000 (USEPA 1993) (Pfaff, 1993). 2.5 Soil sampling and characterization An uncontaminated sandy soil sample was collected from the surface layer (0-10cm depth) from a fallow nursery at Pacific Regeneration Technologies (PRT) Red Rock, Prince George, B.C in Canada. The sample was air-dried for 1 week, thoroughly mixed and passed through a 2-mm mesh sieve. Routine soil analysis was carried out to determine soil textural class, and the fractions sand, silt and clay were quantified according to pipet method (Kalra & Maynard, 1991). 2.5.1 Physical Analysis Saturated paste of some soil sample was prepared by weighing some grams of the sample into a beaker and adding some distilled water as the suspension medium (while stirring with a spatula) until the soil was fully saturated. When the paste slide freely off the spatula and the mixture reflected some light (shiny), it was then left to stand for 24hr. Filter paper was placed inside the Buchner funnel after which the paste was then transferred in. The vacuum pump was opened to allow filtration and was immediately closed when the soil began to crack. Figure 3. Soil saturation: Buchner funnel was used to extract soil water after 24hr 14 The filtrate was transferred into a beaker and the soil pH and electrical conductivity were determined. Carbon and nitrogen were determined using the CHNS elemental analyzer. Other nutrients were also analyzed by the ICP using the aqua regia method. Concentrations of the exchangeable bases: calcium (Ca2+), magnesium (Mg2+), potassium (K+) and sodium (Na+) were extracted with ammonium acetate and analyzed by ICP-OES. From these the cationexchange capacity (CEC) was calculated (Brix, n.d.). The physicochemical properties of soil are shown in table 3 below. Table 3. Physiochemical properties of the soil (n=1) Parameters Unit Value Sand % 95.0 Silt % 2.7 Clay % 2.3 pH - 6.4 EC µs cm-1 78 Organic C % 0.24 Total N % 0.02 CEC cmol/kg 3.0 MC % 21.41 Ca mg/kg 89.10 Mg2+ mg/kg 0.96 K+ mg/kg 7.23 Na+ mg/kg 2.28 Al3+ mg/kg N.D Mn mg/kg 0.42 Fe mg/kg 0.01 N.D.: not detected; CEC: cation exchange capacity. 15 2.6 Greenhouse Pot Experiment The greenhouse experiment was conducted in pod A planting section of the EFL which consisted of a randomized complete block experimental design using a factorial arrangement of treatments. The experiment consisted of six treatments and four replicates each which resulted in a total of twenty-four pots. The compost/soil mixtures were prepared with a 25% compost mass ratio to 75% sandy soil except for the control (S) which consisted of 100% soil. There were 4 seeds of the barley (Hordeum vulgare) sown in each pot (height:15cm and inner diameter:10.5cm) in a completely randomized design and thinned to two after 14 days. Table 4 shows the various planting treatments. During the experiment, all the plants were irrigated daily (manually by weighing each pot) using DI water. Phosphorus, potassium, and nitrogen are totally unavoidable for sustainability in agriculture. They have proven to enhance soil fertility and increase crop productivity. After 4 weeks, water soluble fertilizer (brand: TUNE UP) with chelated trace elements containing: nitrogen (20.0%), available phosphoric acid (P 2O5) (20.0%), soluble Potash (K2O) (20.0%), chelated magnesium (0.04%), chelated Iron (0.04%), chelated manganese (0.03%), chelated zinc (0.02%), boron (0.02%), chelated copper (0.01%), molybdate (0.001%), Cobalt (0.001%), vitamin B (0.0001%) was added as supplements equally to all twenty-four pots as they were seen to be deficient in these nutrients. Application rates were according to recommended rates for barley (Robertson & Stark, 1993). The plants were harvested after 60 days of planting. 16 Table 4. Planting soil treatments Abbreviations Treatments S Soil SC Soil + Compost SCNZ10 Soil + Compost + Natural Zeolite (10%) SCNZ15 Soil + Compost + Natural Zeolite (15%) SCMZ10 Soil + Compost + Modified Zeolite (10%) SCMZ15 Soil + Compost + Modified Zeolite (15%) Added on mass basis: 75% Soil + 25% Compost except for control which had 100% Soil 2.6.1 Plant Growth Data At the end of the experiment, the plants harvested were carefully separated (partitioned) into the shoots, roots and soil. The roots were separated then thoroughly rinsed several times using UNBC tap water (rinsed afterwards once with DI water). The shoots were gently rid of soil or compost particles and the shoot height was measured using a meter rule (from crown to apex) for all treatments. Both the shoots and roots were placed in paper bags and dried in the oven at 55ºC for 72hr. The shoot and root dry weight were determined as shoot and root biomass were recorded per treatment by using a weighing balance. 2.6.2 Shoot and Root Nutrient Content Analyses A coffee blender was used to granulate the root and shoot samples for further analysis such as elemental analysis using the CHNS analyzer, and the metal concentrations (P, K, Ca, Mg, Fe, Mn, Zn, Cu, S, Mn and others) were analyzed by the inductively coupled plasma optical emission spectrometry (ICP-OES). The representative samples were taken from soil mixtures and oven dried at 55ºC for 72hr and analyzed for nutrient concentrations using ICP method. The macronutrients and micronutrients were expressed in mg/kg while the C and N were expressed in % dw. 17 2.7 Statistical Analyses The data were analyzed using 1-way analysis of variance (ANOVA). Comparison of means between treatments by the least significant difference (LSD) test were performed to evaluate the statistical significance of the impact of the addition of different levels and types of zeolite to the various treatments at a significance level of P≤0.05 (SPSS 20.0). The mean values followed by the same letters are not significantly different (p < 0.05). 18 3. Results and Discussions 3.1 Elemental Composition of Natural Zeolite 3.1.1 XRD Analysis The particle size of the clinoptilolite zeolite was ground to powder form and X-Ray diffraction technique (XRD) was used to analyze the zeolite samples. Fig 4. shows the X-ray analysis of both natural and modified zeolites. The X-ray diffraction pattern of both the natural and modified forms of zeolite were the same which shows that the crystalline (structural) framework of the natural zeolite remained unchanged (intact) after the modification process (Soudejani et al., 2018). Figure 4. XRD analysis for NZ (Natural Zeolite) and MZ (Modified Zeolite) 3.1.2 XRF analysis The results of both natural and modified zeolite are given in Table 5. Based on the results, Al and Si compounds constitute the major percentage of both zeolite forms. The concentration of heavy metals in the analyzed compost samples were not significant, this confirms that it is environmentally safe for application (results found in the appendix section) according to BC 19 Organic matter recycling regulation (OMRR). The 0.408% of MgO in the CMZ indicates that Mg is attached to the zeolite samples during the modification process. Table 5. The XRF analysis of NZ (natural zeolite) and MZ (modified zeolite) (n=1) Compounds NZ MZ (%) Al2O3 9.76 9.98 SiO2 61.9 63.5 P2O5 ND 0.0862 SO3 0.0512 0.0516 Cl 0.0408 0.0358 K2O 2.93 3.05 CaO 1.2 1.11 MnO 0.026 0.0255 Fe2O3 1.26 1.23 NiO 0.02 0.022 CuO 0.0093 0.0102 ZnO 0.0169 0.0166 As2O3 0.0006 0.0005 Co2O3 0.0077 0.0095 Rb2O 0.0111 0.0119 SrO 0.0138 0.0149 Y2O3 0.0044 0.0048 ZrO2 0.0233 0.0246 BaO 0.0222 0.0307 PbO 0.0027 0.0032 MgO ND 0.408 Hg ND ND Cr2O3 ND ND U ND ND Bi ND ND Na ND ND As 0.0006 0.0005 Mo ND ND Cd 0.0015 0.0014 Values lower than detection limit were expressed as ND. 20 3.2 Change in Temperature Figure 5 shows the temperature profiles in control and all the zeolite treatments during the composting process. The thermophilic stage is important for the efficient removal of pathogens to generate compost hygienic enough for use (Chan et al., 2016). This was extremely low in comparison to some other studies. The low temperature was attributed to the reactor size, volume of feedstock used to conduct this study and the temperature-controlled chambers in which the reactors were placed throughout the composting process. C CNZ10 CNZ15 CMZ10 CMZ15 39 38.5 Temperature 38 37.5 37 36.5 36 35.5 35 34.5 0 2 4 6 8 10 12 Time (Days) Figure 5. Variation in temperature during the first ten days of composting C- compost + 0% zeolite (control), CNZ10- compost + 10% natural zeolite, CNZ15- compost + 15% natural zeolite, CMZ10- compost + 10% modified zeolite, CMZ15- compost + 15% modified zeolite Within the first week of the composting process, the temperature ranged between 35.0 - 38.5ºC. Lowest temperature was particularly observed in the control treatment (C) which also had the highest moisture content which could have generated an anaerobic condition leading to an unconducive composting environment. The high water retention capacity of zeolite as a result of high porosity, allowed the zeolite treated coils to capture excess moisture and provided more conducive aerobic conditions for the uptake to the degrading microbes (Jiwan et al., 2013). This enhanced microbial activities that helped to degrade the organic matter efficiently as a 21 result of rapid heat production (He et al., 2013). The control treatment with a higher organic matter compared to the other treatments signifies lower organic matter degradation showing a high percentage of unavailable organic matter (Waqas et al., 2019). 3.3 pH Variation pH is a very vital parameter which influences the microbial activities during composting process (Awasthi, Pandey, et al., 2016). The pH values began to drop after day 14 of the composting process because of the formation of organic acids during the organic matter decomposition (Waqas et al., 2019) and eventually goes above neutral because of acid consumption (Figure 6). High concentration of zeolite contributed to the acidity of the compost and caused a decrease in pH. The pH of the treatments was mostly alkaline to neutral throughout the composting process. pH is a good indicator of any soil amendment quality and nutrients within the manure is easily bioavailable when the pH is neutral (Ravindran et al., 2017). The zeolite concentration did not initially have any effect on the different treatments until day 28 when both CNZ treatments reduced to a lower value of 7.4 while the CMZ treatments remained high at 7.7 and 8 respectively. The control was however still the highest at 8.3. The pH value for the control treatment was maintained in the same range from beginning till the end of the experiment meanwhile the presence of zeolite enhanced fluctuations through the composting days as seen above. The final pH values at the end of the composting process were 8.3 for control trial, 7.5 for CNZ10, 7.8 FOR CNZ15, 8.1 for CMZ10 and 7.7 for CMZ15. pH within the range of 7 - 8 is considered to be optimum for composting (Ivankin et al., 2014). This indicates that the maximum and minimum pH were the C (control) and CMZ15 (15% modified zeolite) treatments respectively. Venglovsky et al., 2005 reported that the pH values of the zeolite amended treatments were lower than that of the pig slurry control treatment and this was attributed to the adsorption of ammonium by the zeolite. The pH buffering function of zeolite is still ambiguous as it is dependent on the presence of different soluble ions in the 22 system in the process of slightly reducing the ammonium-based buffering in the composting mass. The high dosage of zeolite could affect its adsorption capacity resulting in dilution effect that affects the pH value and cause the different values under a changing pH condition (Chan et al., 2016) . According to the analysis of variance, the ratio and type of zeolite (natural and modified) in the compost affected these results. 9 C CNZ10 CNZ15 CMZ10 CMZ15 8 7 pH 6 5 4 3 2 1 0 0 14 28 44 60 Time (Days) Figure 6. Changes in pH during the composting process. C- compost + 0% zeolite (control), CNZ10- compost + 10% natural zeolite, CNZ15- compost + 15% natural zeolite, CMZ10compost + 10% modified zeolite, CMZ15- compost + 15% modified zeolite 3.4 Electrical Conductivity (EC) High electrical conductivities signifies high salt concentrations and can result in disruption of soil physical properties, osmotic stress and ion toxicity, which can influence crop germination and most physiological processes in the plant (Cho et al., 2017; Mufwanzala & Dikinya, 2010). Generally, compost possessing high EC are expected to release excess soluble salts which have adverse effect on plant yield and growth. EC measures the total ion concentrations as well as changes in the inorganic levels of ions like Na+ , SO42- , K+ , NO3- , Cl- and NH4+ during the composting process (Waqas et al., 2018; J. Zhang et al., 2014). The EC of all treatments ranged 23 between 2.2 to 2.3 dS/m because of the different composition of the various treatments (Figure 7). While the control always had the highest EC, the modified zeolite had the lowest EC. In the control treatment, EC increased slowly then decreased towards the end of composting because of slow degradation. The natural zeolite treatments also showed a decline in the EC value (1.58 dS/m) at the end of the process. Addition of modified zeolite reduced the EC of the composting mass to 1.38 dS/m which was proportional to the rate of zeolite application. As a result of the high cation exchange capacity of zeolite and its molecular structure, it is able to exchange ions freely on the surface and adsorb ions resulting in a decrease of the EC (Hedström, 2001). The ideal compost should have electrical conductivity no higher than 2 dS/m (Petrik, 1985). 3 C CNZ10 CNZ15 CMZ10 CMZ15 2.5 EC (dS/m) 2 1.5 1 0.5 0 0 14 28 44 60 Time (days) Figure 7. Changes in EC during the composting process. C- compost + 0% zeolite (control), CNZ10- compost + 10% natural zeolite, CNZ15- compost + 15% natural zeolite, CMZ10compost + 10% modified zeolite, CMZ15- compost + 15% modified zeolite. 24 3.5 Final total carbon (C), total nitrogen (N) and C/N ratio for all treatments As shown (figure 8), the total carbon contents in all the treatment decreased at the end of the composting process. The initial C concentrations are found in table A3 of the appendix section. An ideal mature compost should have a total C content of 8 to 35% (Gamze Turan & Nuri Ergun, 2007). Therefore, the compost generated from this research falls within the acceptable range. The lowest percent decrease in organic carbon degradation was observed in the control C (23.7%) < CNZ10 (25.7%) < CMZ10 (25.9%) < CMZ15 (26.3%) < CNZ15 (26.4%) showing that increasing the zeolite dosage caused higher reduction in the carbon content. The total organic carbon content decreased during the composting process as a result of its mineralization by the microorganism as their energy sources (J. W. Wong & Fang, 2000). CNZ10 and CMZ10 have similarities while the CNZ15 and CMZ15 also had similar carbon content. The readily available C was used initially and during the composting process, the decreased metabolic activity resulted in the decrease of available C. The total N content decreased in all treatments especially the control, the percentage decrease is as follows: C (18.8%) < CNZ10 (16.1%) < CNZ15 (15.8%) < CMZ10 (15.6%) < CMZ15 (14.6%). The initial and final concentrations are found in table A3 of the appendix section. There was higher decrease in total N of the control treatment than in the zeolite treatments which was significant according to ANOVA table (found in the appendix section table A). NH4-N content causes reduction in the N content and some undesirable odor that may be increased during the composting as a result of the anaerobic conditions at higher pH levels (Gamze Turan & Nuri Ergun, 2007; J. Wang et al., 2014). During composting, carbon and nitrogen are two very vital nutrients primarily used by microorganisms for production of energy and cell growth. This explains the variability in the C/N ratio which is generally used as an assessment of the maturity of the end product (Awasthi, 25 Wang, Pandey, et al., 2017). The initial C/N ratio across all treatments was higher than the final C/N ratio showing that there was decrease in all the five treatments including the control treatment (shown in table A3 of the appendix section). The lowest final C/N ratio was detected in the CMZ15 (14.9) < CNZ15 (15.0) < CMZ10 (15.1) < CNZ10 (15.2) while the control treatment C had the highest C/N ratio (16.4). A C/N ratio of below 15 in manures is acceptable for direct application to soil but a C/N ratio above this may lead to N immobilization if applied directly to the soil (M. P. Bernal et al., 2009). Based on this, the CNZ15 and CMZ15 treatments are the most applicable to soil however for the purpose of this study, all treatments were used for the planting. The decreasing trend of the C/N ratio was most likely because of the degradation process. The percent decrease between the final and initial C/N ratio across all treatments is as follows: C (6.0%) < CNZ10 (11.5%) < CMZ10 (12.2%) < CNZ15 (12.5%) < CMZ15 (13.6%). This signifies that the higher the zeolite dose the higher the decrease between the final and initial C/N ratio. The total carbon content was reduced because of the microorganisms that mineralize the organic carbon as their energy sources during the composting process. At the end of the composting process, the mass of the produced compost reduced because of the organic decomposition however, the nitrogen was not consumed like the carbon. The highest was observed in CNZ15 and CMZ15 treatments. The weight of the generated compost is summarized in table 6. Increasing the zeolite ratio significantly enhanced the amount of compost generated as well as total N which is nutritional for the plants. Like the C/N, the CNZ15 and CMZ15 treatments had the largest weight which was significantly different from the other treatments. 26 35 Total C (%) 30 a b 25 b c c 20 15 10 5 0 C CNZ10 CNZ15 CMZ10 CMZ15 Total N (%) Treatments 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 a C b CNZ10 c CNZ15 b CMZ10 bc CMZ15 Treatments 17.5 a 17 C/N Ratio 16.5 16 b 15.5 b b 15 b 14.5 14 13.5 13 C CNZ10 CNZ15 CMZ10 CMZ15 Treatments Figure 8. Final C, total N and C/N Ratio where C: compost with no zeolite amendment, CNZ10: compost + 10% natural zeolite, CNZ15: compost + 15% natural zeolite, CMZ10: compost + 10% modified zeolite, CMZ15: compost + 15% modified zeolite. The bar charts represent the means of four replicates (n = 3). Different letters above the error bars indicate statistically significant differences among the six treatments according to LSD test for p<0.05. 27 The control treatment decreased by 35% from its starting weight (1kg), CNZ10 and CMZ10 decreased by 30% and 31.7% respectively from their starting weight (1.1kg) while CNZ15 and CMZ15 decreased by 23.7% and 22.3% respectively from their starting weight (1.15kg). Table 6. Properties of compost produced after 60 days Treatments Carbon (%) C CNZ10 CNZ15 CMZ10 CMZ15 029.0a 23.8b 21.5c 23.6b 21.9c Nitrogen (%) 1.76a 1.56b 1.43c 1.56b 1.46bc C/N ratio 16.43a 15.21b 15.00b 15.09b 14.93b Weight of final compost (g) 650.9a 761.7b 877.6c 751.6b 893.3c 3.6 The effect of natural and modified zeolite on Nitrate Concentration The amount of NO3-N increased at the end of the composting process for all the five treatments including control as shown in figure 9. As seen from the graph, the amount of zeolite added had significant effect on the final concentration compared to the control treatment which had no zeolite in it. Ammonium was produced at the early stage due to the initial breakdown of nitrogenous compounds (Awasthi, Wang, Pandey, et al., 2017; J. W.-C. Wong et al., 2009). The ANOVA results showed that the natural and modified zeolites had significant effect on the ammonium content. The maximum NH 4+ was observed for CMZ15 and CNZ15. Higher adsorption and cation exchange capacity which attracted the NH4+ ions and prevented its conversion to free ammonia (NH3) were contributing mechanisms for why both treatments had the highest values compared to the others (Waqas et al., 2019). 28 NO3 – N (mg/kg) 6000 a 5000 b 4000 c d CNZ15 CMZ10 c 3000 2000 1000 0 C CNZ10 CMZ15 Treatments Figure 9. NO3-N concentration after composting. C: compost with no zeolite amendment, CNZ10: compost + 10% natural zeolite, CNZ15: compost + 15% natural zeolite, CMZ10: compost + 10% modified zeolite, CMZ15: compost + 15% modified zeolite. NH4+ losses in the form of ammonia contributes largely to environmental pollution and also reduces the value of the composted end product (S. Wang & Zeng, 2018). Previous years ago, research studies have confirmed the potential use of zeolites to improve degradation rate, NH4+ adsorption as well as reduction of NH3 losses during the process of composting (Chan et al., 2016; Jiwan et al., 2013). The higher the zeolite ratio, the more reduction in nitrate production. Comparing the initial NO3-N concentration to the final concentrations, it is evident that the nitrate production was minimal in CNZ15 and CMZ15 (15.56% and 11.29% respectively) followed by CNZ10 and CMZ10 (26.27% and 21.13% respectively). However, on the other hand, the control had the maximum NO3-N production (45.48%). 3.7 The effect of natural and modified zeolite on NH4-N Figure 10 shows the result that the amount and type of zeolite affected the NH4-N content in the compost. The natural zeolite surprisingly had low concentration (69.1mg/kg) while the NH4 concentration for control treatment was127.2mg/kg. The maximum concentration was observed in CMZ15 (190.7mg/kg) > CMZ10 (152.5mg/kg) > CNZ15 (134.4mg/kg). Mechanisms such as high adsorption and cation exchange capacity were responsible for 29 modified zeolite having the maximum concentration (Waqas et al., 2019). Over the past couple of years, research studies have reported the potential use of zeolites to improve degradation rate, adsorption of ammonium and reduce losses during the composting process (Chan et al., 2016; Jiwan et al., 2013). It was also observed (data not shown) that the moisture content retained in samples with zeolite were higher than that of the control treatment without zeolite addition. NH4 - N (mg/kg) 250 d 200 150 a a c b 100 50 0 C CNZ10 CNZ15 CMZ10 CMZ15 Treatments Figure 10. NH4 -N Concentration. C: compost with no zeolite amendment, CNZ10: compost + 10% natural zeolite, CNZ15: compost + 15% natural zeolite, CMZ10: compost + 10% modified zeolite, CMZ15: compost + 15% modified zeolite. The concentration of P, Mg, Na, and Cu were higher in the zeolite treatments compared to the control treatments while the concentrations of Ca, Mn, K, Fe, Zn and S were lower in the zeolite treatments compared to the control treatments in the final compost. It is significant to note that most of the heavy metals (analyzed by acid digestion through the ICP-OES)) had low values (within acceptable limits) across the zeolite treatments and were in accordance to the British Columbia organic matter recycling regulations (MWLAP, 2002) (Columbia, 2002). The change in concentration of the metals is dependent on the metal loss through leaching and the total concentration of metals as a result of organic matter destruction (Wagner et al., 1990). 30 Table 7. Mehlich-3 concentration of final compost Metals C CNZ10 CNZ15 CMZ10 CMZ15 16140.1±402.6 Ca 18915.7±137.2 17199.8±557.1 15736.8±477.5 15153.2±422.8 Mg Mn 246.25±27.1 266.87±2.2 2551.02±63.58 2539.81±51.17 278.56±16.48 2529.1±53.81 265.26±5.76 263.51±5.66 2455.26±172.55 2352.95±37.64 Na P S 0.77±0.06 0.73±0.15 2278.37±117.3 1.43±0.08 2.06±0.04 1557.65±2.7 2.42±0.06 2.84±0.08 1508.15±20.6 Zn Cu Fe 300.94±2.2 259.03±6.7 67.61±5.08 77.50±7.21 19213.3±680.4 15063.6±292.9 227.49±9.6 99.99±11.5 14087±470 225.9±0.8 230.17±6.16 106.14±7.07 104.39±11.2 12468.1±1176.4 13370.2±315.6 K 4110.27±112.8 3362.06±91.9 3596.51±237.9 3323.59±93.4 2.07±0.08 1.97±0.007 1606.2±59.3 1.48±0.29 2.92±0.1 1268.1±23.5 3953.95±28.8 ICP-OES final mehlich-3 concentration of metals (mg/kg) for final compost showing the mean and standard deviation of all the treatments (n=3). C: compost with no zeolite amendment, CNZ10: compost + 10% natural zeolite, CNZ15: compost + 15% natural zeolite, CMZ10: compost + 10% modified zeolite, CMZ15: compost + 15% modified zeolite. 3.8 Effect of natural and modified zeolite on mehlich-3 extractable final P concentration of the compost after 60 days: This is one of the main objectives of the present study hence it was quite interesting to report the significant differences between the compost without zeolite treatment and compost with natural and modified zeolite treatments. From table 7, CNZ15 and CMZ15 retained the highest P concentrations in comparison to the control and CNZ10 and CMZ10 treatments. The order is as follows: CMZ15 (2.9mg/kg) > CNZ15 (2.8mg/kg) > CNZ10 (2.0mg/kg) > CMZ10 (1.97mg/kg) > C (0.7mg/kg). 31 Final P Conc. (mg/kg) 3.5 2.5 b 2 1.5 1 c c 3 b a 0.5 0 C CNZ10 CNZ15 CMZ10 CMZ15 Treatments Figure 11. Concentration of mehlich-3 Phosphorus (P) in the final compost after 60 days of composting. C: Compost; CNZ10: Compost+Natural Zeolite (10%); CNZ15: Compost+Natural Zeolite (15%); CMZ10: Compost +Modified Zeolite (10%); CMZ15: Compost +Modified Zeolite (15%) The bar charts represent the means of three replicates (n = 3). Different letters above the error bars (standard deviation of the mean values) indicate statistically significant differences among the five treatments according to LSD test for p<0.05. Modified and natural zeolites were able to trap the P unlike in the control treatment where very minimal amount was retained after the composting process was completed. The initial mehlich3 concentration of phosphorus was much higher as shown in table A7 in the appendix section. The total P content in chicken manure is usually high as a result of the fact that the chicken only make use of minimal portion of ingested P from their feed while the rest is being excreted (G. Li et al., 2014). 3.9 Shoot Growth The compost and zeolite treatments showed significantly higher plant height and biomass in comparison to the control treatments. Figure 12 shows the difference among all the treatments for shoot height and biomass. The highest shoot height was observed in the SCMZ15 (58.8cm) > SC (58.7cm) > (57.1cm) > SCMZ10 (54.3cm) > SCNZ15 (51.6cm) while the control: S was the least in height (43.3cm). There were no significant differences between the SC and the zeolite treatments as well as between the natural and modified zeolites treatments except for SCNZ15 and SCMZ10. The shoot largest biomass was found in SCMZ15 (7.67g/kg) 32 >SCMZ10 (7.24g/kg) > SCNZ15 (6.38g/kg) > SCNZ10 (6.19g/kg) > SC treatment (5.99g/kg) > S (0.48g/kg). There were no significant differences observed between the compost and zeolite treatments except for SCMZ10 and SCMZ15 which were also significantly greater than the natural zeolite treatments. This shows that the barley crop responded positively to the zeolite amendment especially for the 15% modified zeolite treatment because it yielded the largest biomass and had the highest height compared to the other treatments. This improvement in growth in comparison to the others can be attributed to the essential nutrients contained in Shoot height (cm) zeolite (Al-Busaidi et al., 2008). 70 60 50 40 30 20 10 0 b b SC SCNZ10 a S b c d SCNZ15 SCMZ10 SCMZ15 c c Shoot biomass (g/kg) Treatments 10 8 b 6 b b SCNZ10 SCNZ15 4 2 0 a S SC SCMZ10 SCMZ15 Treatments Figure 12. The shoot height and shoot biomass for different treatments: S: Soil; SC: Soil+Compost; SCNZ10: Soil+Compost+Natural Zeolite (10%); SCNZ15: Soil+Compost+Natural Zeolite (15%); SCMZ10: Soil+Compost+ModifiedZeolite(10%); SCMZ15:Soil+Compost+ModifiedZeolite (15%). The bar charts represent the means of four replicates (n = 4). Different letters above the error bars indicate statistically significant differences among the six treatments according to LSD test for p<0.05. 33 It was reported in the past that zeolite application increased cation exchange ability, water retention and plant nutrients (Ayan et al., 2005). The low dry matter production for control treatment was due to lack of sufficient fertilization to sustain plant growth and development (Nur Aainaa et al., 2018). The zeolite treatments were able to retain enough nutrients and timely release them as they are necessary for plant growth and development. Low dry matter for control treatment was as a result of no fertilization to sustain growth and development (Hasbullah et al., 2014). Previous reports showed that addition of a compost containing natural and mg-modified zeolite to soil increased the shoot dry weight of corn by 40% and 56% respectively (Soudejani et al., 2019). Figure 13 shows representative pots of the various treatments at the day of harvesting. SC S SCNZ15 SCMZ10 SCNZ10 SCMZ15 34 Figure 13. Pictorial representation of all the six different treatments after 45 days of planting. S: Soil; SC: Soil+Compost; SCNZ10: Soil+Compost+Natural Zeolite (10%); SCNZ15: Soil+Compost+Natural Zeolite (15%); SCMZ10: Soil+Compost+ModifiedZeolite (10%); SCMZ15: Soil+Compost+ModifiedZeolite (15%) 3.9.1 Shoot Nutrient Analysis Nitrogen (N) Significantly lower concentration of N was observed in other treatments when compared to the control treatment; S (4.3%) because of dilution effect as shown in figure 14. The lowest N concentration was observed in SCMZ10 and SCNZ15 (both 2.6%) < SC (2.7%) < SCNZ10 < (2.8%) < SCMZ15 (3.0%). Similar findings were observed when zeolite was combined with manure and compared with control soil treatment (Chatzistathis et al., 2021). There were no significant differences between the SC and zeolite treatments except for SCMZ15. This implies that the SCMZ15 treatment despite having the best plant yield was still able to have the highest N content compared to the other treatments. Table A4 in the appendix section shows the shoot nutrient concentration for all treatments. Figure 14 shows the graphical representation of shoot concentration of nutrients of all the treatments. Another likely explanation for the low N content in the other treatments besides the control treatment could be the co-effect of increased N microbial immobilization or heterotrophic denitrification processes caused by the organic C and high availability of nitrate in these treatments(Burger & Jackson, 2003; Calderón et al., 2004). As N is a vital plant nutrient responsible for protein and chlorophyll chemical structures. The plant N and P content were significantly lower across the compost and zeolite treatments for plants and roots compared to the control treatments because of its use of the nutrients for improved yield or growth. The difference between these treatments can be explained by nitrate leaching and plant uptake. Little improvement in the status of N was observed in soils from Texas that were treated with zeolites (Mumpton, 1999) while in Japan and Taiwan, there was 35 significant improvement in the amount of available nitrogen in sandy and paddy soils respectively after adding 90 ton zeolites per ha (Hsu et al., 1967; Minato, 1968b). The zeolites have the capacity to adsorb the N from the compost in the form of NH 4-N and release it eventually as a slow-releasing fertilizer (Taheri‐Soudejani et al., 2019). Phosphorus (P) P is an essential and often limiting nutrient for plant growth (Hirsch et al., 2006). All the compost and zeolite treatments showed significant decrease in shoot P concentration in comparison to the control treatment; S (7400mg/kg) except for SCNZ10 treatment (7448mg/kg) which was not significantly different from the control treatment. The lowest P concentration was observed in SCMZ10 treatment (6300mg/kg) < SCNZ15 (6500mg/kg) < SCMZ15 and SC treatment (both had 6700mg/kg). There were no significant differences observed between the SC and zeolite treatments except SCNZ10 and SCMZ10. Between the natural and modified zeolites, there were no significant differences except for SCNZ10. The modified zeolites captured the P and as a slow releaser, it retained the P in the soil and releases as needed to the plants and roots tissues. The P content was observed to be significantly highest in the soil mixture in comparison to the plants and roots. Similar results were observed in Solanum Lycopersicon L. plants where the control treatment had significantly higher P content compared to the manure + zeolite treatment and were the best in regards to P fertilization (Chatzistathis et al., 2021). P and K similarity can be explained by ion exchange reactions of K and strong nonspecific sorption of P (Loganathan et al., 2014) . This helps to minimize leaching of both nutrients as much as possible. 36 Potassium (K) Unlike Ca and Mg, zeolite addition did not significantly affect monovalent K concentration because its availability is mostly regulated by soil properties and plants requirements. The compost and zeolite treatments showed significant increase in concentration of K in the plants in comparison to the control treatment; S (3300mg/kg) except for SCNZ10 (3200mg/kg). The highest K concentration was observed in SC treatment (4100mg/kg) > SCNZ15 (3900mg/kg) > SCMZ15 (3800mg/kg) > SCMZ10 treatment (3600mg/kg). There were no significant differences between the SC and zeolite treatments except for SCNZ10 and SCMZ10. For the natural and modified zeolites, there were no significant differences detected except for SCNZ10. Available K concentration was higher in the compost treatments compared with either the zeolite or control treatments. It was however not significantly different from SCMZ15 and SCNZ15 treatments which shows that the effect of plant uptake on the K concentrations were very minimal in comparison to Mg and Ca. As a result of the combined high concentration of K in the compost and zeolite which clearly boosted K uptake in the compost and zeolite treatments, significantly higher K content was observed. Also, the application of the NPK fertilizer added was a contributing factor. This also shows how beneficial the role of zeolite is in enhancing K uptake by the barley plants. Zeolites are able to act as a macro nutrient supplement (Jakkula & Wani, 2018). This result was in line with the research conducted to investigate the significant increase in K uptake after zeolite addition for pepper plants (Assimakopoulou et al., 2020) and also tomato plants (Doostikhah et al., 2020). Addition of zeolite to soils is significant and it does not have any influence on soil humus content and it’s composition (Filcheva & Tsadilas, 2002). These observations clearly show the beneficial role of zeolite which is similar in achieving good results using inorganic fertilization in improving K uptake by Solanum Lycopersicon L. plants (Chatzistathis et al., 2021). However, excess K reduced Mg and Ca concentrations in the plants, hence when the former is 37 in abundance then the latter got reduced. This is the reason why they were not as high in zeolite treatments in the plant shoots of the Hordeum vulgare because there is already high concentration of K present. Zeolite also had K accumulation because of its retention capacity of K and NH4+. K concentration was highest in the compost treatment compared to others meaning that its K concentration was sufficient for plant growth and as a result the effect of plant uptake was minimal. 5 a Total N (%) 4 b 3 b bc c b 2 1 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 10000 a Ca(mg/kg) 8000 6000 4000 b 2000 0 S SC c cd c d SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 38 7000 a Mg (mg/kg) 6000 5000 4000 cd c 3000 2000 d c b 1000 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 50000 K (mg/kg) 40000 b bc a a c bc 30000 20000 10000 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 9000 8000 a P (mg/kg) 7000 b a bc c b 6000 5000 4000 3000 2000 1000 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 39 Fe (mg/kg) 200 180 160 140 120 100 80 60 40 20 0 a S b b b b b SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 d d d SCNZ15 SCMZ10 SCMZ15 Treatments 140 a Mn (mg/kg) 120 100 b 80 60 c 40 20 0 S SC SCNZ10 Treatments 120 Cu (mg/kg) 100 a b 80 c bc b d 60 40 20 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 40 120 b Zn (mg/kg) 100 b c 80 c c 60 40 a 20 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 S (mg/kg) Treatments 4000 3500 3000 2500 2000 1500 1000 500 0 b b c a S SC SCNZ10 c c SCNZ15 SCMZ10 SCMZ15 Treatments Figure 14. Shoot concentration of nutrients among the treatments. Means with similar letters are not significantly different (p < 0.05) Calcium (Ca) and Magnesium (Mg) Total Ca and Mg contents were statistically similar across all the treatments. The compost and zeolite treatments had significantly lower plant concentration of total Ca and Mg because of dilution effect in comparison to the control treatments: S (8800mg/kg and 5700mg/kg respectively). Similar results were reported when control soil was compared to manure mixed with zeolite treatments (Chatzistathis et al., 2021). The lowest Ca concentration was observed in the SC treatment (1800mg/kg) < SCMZ10 (2400mg/kg) < SCNZ10 (2500mg/kg) < SCNZ15 41 (30000mg/kg) < SCMZ15 (3200mg/kg). Similarly, the lowest Mg concentration was observed in SC treatment (1800mg) < SCNZ10 and SCMZ10 treatments (both 2300mg/kg) < SCNZ15 (2400mg/kg) < SCMZ15 (2700mg/kg). There were significant differences between the SC and zeolite treatments for both nutrients (Ca and Mg) but no significant differences between the natural and modified zeolites. The highest Mg and Ca plant concentrations were found in the control (S) treatments because of its low plant yield while other treatments with higher plant yield had lower concentration of Mg and Ca nutrients. The compost and zeolite adsorbed the Mg and Ca because of high crop yield and growth during the planting process hence lower concentration in comparison to the control treatment. Highest Ca concentration was however detected in the SCMZ15 treatment despite the dilution effect encountered. SCMZ15 and SCNZ15 however showed the highest concentrations compared to the other treatments which implies that there was better retention of both nutrients in the higher dose of zeolite. Similar observation was reported in tomato cultivated with zeolite (Stylianou et al., 2004). Iron (Fe), Manganese (Mn) and Copper (Cu) Fe, Mn and Cu accumulation was recorded in the control treatment in comparison to the other treatments. There was lower concentration of Fe in the plants of other treatments compared to the control treatment; S (181mg/kg) because of dilution effect. The lowest Fe concentration was observed in SCMZ15 (96.7mg/kg) < SCNZ15 (97.2mg/kg) < SCMZ10 (98.7mg/kg) < SCNZ10 (100mg/kg) < SC (103mg/kg). There were no significant differences between the SC and zeolite treatments as well as between the different zeolite types. Mn concentration in the plants because of dilution factor was lowest in other treatments in comparison to the control treatment; S (126.2mg/kg). The lowest Mn concentration was observed in SCMZ10 (25.1mg/kg) < SCNZ15 (25.5mg/kg/kg) < SCMZ15 (26.3mg/kg) < SCNZ10 (41.3mg/kg) < SC (75.4mg/kg). There were significant differences between the SC and zeolite treatments but 42 no significant difference between the natural and modified zeolites except for SCNZ10. The concentration of Cu in the plants of other treatments in comparison to the control treatment; S (92.8mg/kg) was low. The lowest Cu concentration was observed in SCMZ15 treatment (56.1mg/kg) < SCNZ15 (70.4mg/kg) < SC treatments (70.8mg/kg) < SC treatment (70.8mg/kg) < SCNZ10 (81.5mg/kg). There were significant differences between the SC and zeolite treatments except for SCNZ15 and SCMZ10. Significant differences were observed as well between the natural and modified zeolite treatments except for SCMZ10 that was not significantly different from the two natural zeolite treatments. This could be an indication that application of zeolite could be an optional fertilization strategy for micronutrients (such as Mn, Fe and Cu) as it was able to decrease their high chemical fertilization inputs in the barley plant. Similar findings were recorded for Solanum Lycopersicon L. (Chatzistathis et al., 2021). Zinc (Zn) and Sulphur (S) All the compost and zeolite treatments displayed significant increase in the plant concentration of Zn compared to the control treatment; S (37.3mg/kg). The highest Zn concentrations was observed in SCNZ10 treatment (95.9mg/kg) > SC (87.5mg/kg) > SCNZ15 (71.9mg/kg) > SCMZ10 and SCMZ15 (66.9mg/kg). There were significant differences observed between the SC and zeolite treatments except for SCNZ10 but no significant differences were observed between the natural and modified zeolites except for SCNZ10. The concentration of S in the plants of other treatments were higher in comparison to the control treatment; S (2400mg/kg). The highest S concentrations were observed in SCNZ10 (3600mg/kg) > SC (3400mg/kg) > SCNZ15 (3100mg/kg) > SCMZ15 (3000mg/kg) > SCMZ10 (2900mg/kg). There were significant differences observed between the SC and zeolite treatments except for SCNZ10 but no significant differences were observed between the natural and modified zeolites except for SCNZ10. Research has shown that micronutrients can proceed with other nutrients to form 43 insoluble compounds (become unavailable) for plant uptake (Karamanos, 2013). The application of zeolite significantly increased Zn and S content in the plants and the roots. 3.10 Root Growth: The root biomass increased significantly in the compost and zeolite treatments in comparison to the control treatment; S (0.2g/kg). The highest root biomass was observed in SCMZ15 (1.7g/kg) followed by SCMZ10 (1.5g/kg), SCNZ15 (1.4g/kg), SC (1g/kg) then finally the SCNZ10 (0.9g/kg) treatment. Zeolites are known to retain essential nutrients in the root zone when integrated into the soil which allows these nutrients to be used by plants when required (Méndez-Argüello et al., 2018). The presence of more nutrients in the root zone may lead to higher crop yields (Nakhli et al., 2017). Figure 15 shows the root biomass across all the treatments. There were significant differences between the compost and zeolite treatments except for the SCNZ10 treatment which was not significantly different from the SC. There were significant differences between the natural and modified zeolites except for SCNZ15 and SCMZ10. Root biomass (g/kg) 2 c 1.5 b b SC SCNZ10 cd d 1 0.5 0 a S SCNZ15 SCMZ10 SCMZ15 Treatments Figure 15. The root biomass for different treatments: S: Soil; SC: Soil+Compost; SCNZ10: Soil+Compost+Natural Zeolite (10%); SCNZ15: Soil+Compost+Natural Zeolite (15%); SCMZ10: Soil+Compost+ModifiedZeolite (10%); SCMZ15: Soil+Compost+ModifiedZeolite (15%). The bar charts represent the means of four replicates (n = 4). Different letters above the error bars indicate statistically significant differences among the six treatments according to LSD test for p<0.05. 44 3.10.1 Root Nutrient Analysis: Nitrogen (N) The compost and zeolite treatments showed significantly lower concentration of N in the roots in comparison to the control treatment; S (3.1%). The lowest N concentration was observed in both SC and SCNZ10 (1.2%) < SCNZ15 (1.3%) < SCMZ10 (1.5%) < SCMZ15 (1.8%). There were no significant differences between the SC and the zeolite treatments except for SCMZ10 and SCMZ15 which were also significantly different from the natural zeolite treatments. Phosphorus (P) The concentration of P in the plant decreased significantly in the zeolite treatments in comparison to the compost (SC) and soil (S) treatments. Both the roots of S and SC treatments had the highest P concentration of 3500mg/kg and 3300mg/kg respectively which were significantly different from the other treatments. The lowest P concentrations were observed in SCNZ15 (2600mg/kg) < SCMZ15 (2700mg/kg) < SCMZ10 (2800mg/kg) < SCNZ10 (2900mg/kg). No significant differences were observed between the natural and modified zeolite treatments. Potassium (K) The compost and zeolite treatments showed significant decrease in concentration of K for the roots in comparison to the control treatment; S (16000mg/kg). The highest K concentration was observed in SC treatment (13000mg/kg) followed by SCNZ10 and SCMZ10 treatments (10000mg/kg), SCNZ15 treatment (9400mg/kg) then SCMZ15 treatments (8000mg/kg). There were significant differences between the SC and the zeolite treatments. For the natural and modified zeolites, there were no significant differences detected except for SCMZ15. 45 3.5 a Total N (%) 3 2.5 2 b 1.5 b b c d 1 0.5 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 7000 d 6000 b Ca (mg/kg) 5000 4000 3000 b b c a 2000 1000 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 3000 d Mg (mg/kg) 2500 2000 1500 a bc S SC a ab c 1000 500 0 SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 46 K (mg/kg) 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 a b c S SC c c d SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 4000 3500 a a b P (mg/kg) 3000 b b b 2500 2000 1500 1000 500 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 4500 a 4000 Fe (mg/kg) 3500 3000 2500 c 2000 1500 1000 b 500 0 S SC b b b SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 47 300 Mn (mg/kg) 250 a b 200 150 c 100 d 50 0 S SC d c SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments Cu (mg/kg) 1000 900 800 700 600 500 400 300 200 100 0 bc b d bd c a S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 120 100 Zn (mg/kg) e b bc c 80 d 60 40 a 20 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments 48 3500 b 3000 b S (mg/kg) 2500 c b c 2000 1500 a 1000 500 0 S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 Treatments Figure 16. Root concentration of nutrients among the treatments. The root total carbon and total nitrogen for different treatments: S: Soil; SC: Soil+Compost; SCNZ10: Soil+Compost+Natural Zeolite (10%); SCNZ15: Soil+Compost+Natural Zeolite (15%); SCMZ10: Soil+Compost+ModifiedZeolite (10%); SCMZ15: Soil+Compost+ModifiedZeolite (15%). The bar charts represent the means of four replicates (n = 4). Different letters above the error bars indicate statistically significant differences among the six treatments according to LSD test for p<0.05. Calcium (Ca) and Magnesium (Mg) Mg and Ca behaved similar across all the treatments for both the shoots and roots. In the roots, SCMZ15 and SCMZ10 both had the highest concentrations for Mg and Ca which means that both modified zeolite treatments had the higher capacity to adsorb these nutrients compared to the others especially the control treatment proving zeolite’s power as a slow-release fertilizer. The concentration of Ca in the roots significantly increased in the compost and zeolite treatments compared to the control treatment; S (2900mg/kg). The highest root concentration of Ca was detected in SCMZ15 (6300mg/kg) > SCMZ10 (5000mg/kg) > SC and SCNZ15 (both 4700mg/kg) > SCNZ10 (3800mg/kg) treatments. There were no significant differences between the compost and zeolite treatments and between natural and modified zeolites except for SCNZ10 and SCMZ15. The concentration of Mg in the roots significantly increased in the SC, SCMZ10 and SCMZ15 treatments in comparison to the S, SCNZ10 and SCNZ15 49 treatments. The highest root concentration of Mg was detected in SCMZ15 (2300mg/kg) > SCMZ10 (1700mg/kg) > SC (1500mg/kg). Meanwhile the concentration of Mg in the S, SCNZ10 and SCNZ15 treatments were 1300mg/kg, 1300mg/kg and 1400mg/kg, respectively. There were no significant differences between the compost and zeolite treatments and between natural and modified zeolites except for SCMZ15. Iron (Fe) and Manganese (Mn) The concentration of Fe in the roots significantly decreased in the compost and zeolite treatments because of dilution effect in comparison to the control treatment; S (3800mg/kg). The lowest Fe concentration was observed in SC treatment (500mg/kg) < SCNZ10 (580mg/kg) < SCNZ15 (810mg/kg) < SCMZ10 (970mg/kg) < SCMZ15 (1700mg/kg). However, there were no significant differences between the SC and zeolite treatments as well as between the different zeolite types except for SCMZ15 treatment. The compost and zeolite treatments showed significantly lower concentration of Mn in the roots in comparison to the control treatment; S (244.5mg/kg). The lowest Mn concentrations was observed in SCNZ15 (54.2mg/kg) < SCMZ10 < (64.3mg/kg) < SCMZ15 (80.6mg/kg) < SCNZ10 (85.4mg/kg) < SC (196.3mg/kg). There were significant differences between the SC and the zeolite treatments as well as between the natural and modifies zeolites. Zinc (Zn), Sulphur (S) and Copper (Cu) The concentration of Zn in the roots significantly increased in the compost and zeolite treatments in comparison to the S treatments (33.2mg/kg). The highest root concentration of Zn was detected in SCMZ15 (109.4mg/kg) > SC (90.6mg/kg) > SCMZ10 (79.5mg/kg) > SCNZ10 (77.2mg/kg) > SCNZ15 (57.5mg/kg). There were significant differences between the compost and zeolite treatments except for SCMZ10 treatment which was not significantly different from the SC treatment. There were significant differences between the natural and 50 modified zeolites except for SCNZ10 and SCMZ10 treatments which were not significantly different. The concentration of Sulphur (S) in the roots significantly increased in the compost and zeolite treatments in comparison to the control treatment; S (1200mg/kg). The highest root concentration of Sulphur was detected in SC (2700mg/kg) > SCMZ15 and SCMZ10 (both 2500mg/kg) > SCNZ10 and SCNZ15 (both 2100mg/kg) treatments. There were no significant differences between the compost and zeolite treatments except for SCNZ10 and SCNZ15 treatments. There were significant differences between natural and modified zeolites treatments. The compost and zeolite treatments showed significantly higher concentration of Cu in the roots in comparison to the control treatment; S (200.1mg/kg) The highest Cu value was detected in SCMZ10 (781.4mg/kg) >SC (712.5mg/kg) > SCMZ15 (672.5mg/kg) > SCN15 (595.2mg/kg) > SCNZ10 (457.4mg/kg). There were no significant differences between the SC and zeolite treatments except for SCNZ10 and SCMZ10. Between the natural and modified zeolites treatments, there were significant differences observed except for SCNZ15 and SCMZ15. 51 4. Conclusion and Recommendation Zeolites are eco-friendly, cost effective and a good source for increasing crop yield and reducing nitrate leaching. They can be used as an environmentally friendly substrate to develop slow-release fertilizer. In this study, we were able to prove that natural and modified zeolites have the potential to modify and improve the conventional composting process especially when the inorganic mineral is sufficiently added (the higher the mass/ratio, the better the result/output). For the various parameters it was evident that SCNZ15 and SCMZ15 had similar characteristics and proved the most adequate (effective) in terms of pH normalization, EC stability and the mass of final compost generated. Addition of natural and modified zeolites to the compost decreased the NO3-N production compared to the control treatment thereby preventing nitrification process. With regards to the main objective of this research, at the end of the composting process the higher ratios of the natural and modified zeolites (CNZ15 and CMZ15) were able to retain (capture) P in comparison to the control (C) and lower ratios of the natural and modified zeolites (CNZ10 and CMZ10). This confirmed the hypothesis that zeolite can prevent P leaching into the environment (run-off) hence is a good adsorber of P which helps to reduce environmental pollution such as eutrophication. The treatments SCMZ15, SCNZ15 and SCMZ10 behaved similar in terms of the plant and root nutrient concentrations for virtually all the macronutrients and micronutrients. Besides the control treatment, they had the highest concentration especially for the macronutrients in both the roots and plants compared to the other treatments. This indicates that the higher the zeolite ratio applied, the better the nutrient concentration and plant nutrient uptake which indicates that there was adsorption of those nutrients by the zeolite and there was partial release for plant use in comparison to the control treatment. 52 Bibliography Al-Busaidi, A., Yamamoto, T., Inoue, M., Eneji, A. E., Mori, Y., & Irshad, M. (2008). 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Springer. 63 Appendix Table A1: ICP-OES elemental Composition analysis of Natural and Modified Zeolite (n=2): Trace elements in acid digestate (70%(w/w) HNO 3; 34-37%(w/w) HCl=3:1, digested using digestion blocks (DigiPrep) for 6 hours via automated control of temperatures up to 95ºC. Element Al As B Ba Ca Cd Co Cr Cu Fe Hg K Mg Mn Mo Na Ni P Pb S Sb Se Sn U V Zn Zr Unit mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Natural Zeolite 21580±525.83 <6.75 3±0.09 417±10.52 6583±120.52 <0.23 <0.75 3±0.16 6±0.16 1914±17.74 <3.3 21106±451.79 1187±20.16 36±0.63 <1.05 4960±79.67 3±0.02 57±2.10 9±0.15 73±3.70 <4.1 <9 <6.8 <4 3±0.003 60±0.08 19±0.15 Modified Zeolite 22321±531.98 <6.75 2±0.25 424±13.83 5626±101.59 <0.23 <0.75 3±0.22 6±0.03 2023±219.44 <3.3 21302±806.16 2750±4.71 45±3.29 <1.05 3571±111.20 2±0.05 65±4.72 10±0.81 73±1.48 <4.1 <9 <6.8 <4 3±0.418 59±0.03 19±0.57 64 Table A2: NO3 – N and NH4 – N concentration of final compost Treatments NO 3 – N (mg/kg) NH 4 – N (mg/kg) C CNZ10 CNZ15 CMZ10 CMZ15 5305.2a 3922.8b 3425.2c 3667.0d 3260.2c 127.2a 69.1b 134.4a 152.5c 190.7d Table A3: Initial and final concentrations of N, C , C/N and Compost Mass N (%) C (%) C/N Initial Final Initial Final Initial Final COMPOST MASS (g) Initial Final C 2.18 1.77 38.0 29.00 17.47 16.42 1000.18 650.91 CNZ10 1.87 1.57 32.10 23.83 17.19 15.21 1100.12 761.77 CNZ15 1.70 1.43 29.20 21.50 17.15 15.00 1150.33 877.59 CMZ10 1.86 1.57 31.90 23.63 17.18 15.09 1100.39 751.65 CMZ15 1.72 1.47 29.70 21.90 17.30 14.93 1150.09 893.26 65 K 33699.5a 40893.8b 31948.5a 39303.8bc 36543.8c 38232.2bc P 7390.25a 6712.75b 7448.75a 6501.75bc 6333.75c 6752.25b 8803.5a 1821.5b 2495.0c 2965.8cd 2440.3c 3226.3d Ca Zn (mg/kg) 5708.25a 37.30a 1808.50b 87.52b 2319.00c 95.90b 2391.50cd 71.90c 2303.75c 66.87c 2719.00d 66.92c Mg 126.20a 75.40b 41.30c 25.55d 25.10d 26.30d Mn 4970b 6318d 10380.5c 41.2d 41.92bc 1.8d 2667.7b 8075.7d SCMZ10 SCMZ15 1.5c 2824b 9422.75c 41.77bc 1.3b 2658b SCNZ15 57.7d 2355d 109.4e 1715.5c 79.5bc 4670.2b 1429ab 1272.7a 77.2c 90.6b 1.2b 2942.2b 10408.25c 3868c 1520bc 42.05c 4750b SCNZ10 13500.2b 1.2b 3354a 41.52b SC 1282a Zn 33.2a 2937a Mg 3.1a 3512.2a 16340a Ca 40.42a K S P (mg/kg) N (%dw) Treatments C Cu 80.6c 64.3d 54.2d 85.4c 577.7b 497b 3848a Fe 92.85a 70.87b 81.50c 70.45b 77.10bc 56.10d Cu 974.7b 2459.0a 3393.5b 3568.2b 3088.2c 2985.0c 3030.7c S 2487.2b 2101.7c 2099.2c 2710.7b 1195a S 181.00a 103.00b 100.00b 97.25b 98.75b 96.75b Fe 672.6bd 1732.2c 2532.7b 781.4d 595.2bc 807.2b 457.4c 196.3b 712.5b 244.5a 200.1a Mn Tables A5: Root nutrient (elemental) concentrations for Hordeum vulgare for all treatments. S SC SCNZ10 SCNZ15 SCMZ10 SCMZ15 N (% dw) 40.05a 4.25a 40.58b 2.73b 40.72b 2.78bc 40.50b 2.68b 40.30ab 2.60b 39.85c 3.03c Treatments C Table A4: Plant nutrient(elemental) concentrations for Hordeum vulgare for all treatments. 66 Table A6: Analysis of Variance (ANOVA) for compost experiment Compost parameter F -Value P-Value Final C/N ratio 6.361 0.008 Final N (%) 15.200 0.000 Final C(%) 44.233 0.000 Final Compost weight (g/kg) 306.239 0.000 Final NH4 -N (mg/kg) 62.874 0.000 Final NO3 -N (mg/kg) 149.498 0.000 EC (dS/m) 22.750 0.000 pH 120.750 0.000 Final P (mg/kg) 273.326 0.00 Table A7: Initial mehlich-3 concentration of elements (mg/kg) for final compost showing the mean and standard deviation of all the treatments (n=3) Metals C CNZ10 CNZ15 CMZ10 CMZ15 Ca 37540.7±840.7 34793.4±745.2 30171.7±399.9 32194.4±712.5 30517.5±1184.4 Mg 9470.89±290.12 7731.88±68.8 6526.12±239.8 8129.44±164.97 7432.67±131.6 Mn Na 810.24±1.58 5170.17±119.85 741.31±18.21 5170.88±58.62 666.45±26.83 5096.53±19.78 679.47±15.04 4833.45±190.53 665.39±43.7 4599.14±98.85 P 22229.27±678.9 17623.04±295.5 15011.22±435.4 17335.00±431.2 14679.93±174 S 5137.05±92.4 3923.56±79.1 3179.22±117.9 3768.37±44.4 2448.88±91.9 Zn Cu Fe K 588.44±13.2 19.6±1.2 167.56±1.44 37642.2±2019.2 515.98±840 25.62±1.9 255.74±21.2 31319.3±754.6 447.44±12 31.91±5.43 263.13±3.1 27015±571.6 513.89±4.84 78.56±17.96 185.64±8 31379.3±244.7 437.65±6.2 53.01±5.87 230.76±10 26236±692.5 67 Table A8: Total elemental analysis for the final compost of all treatments (mg/kg) showing the mean values and standard deviation (n=3) Elements Al As B Ba Ca Cd Co Cr Cu Fe Hg K Mg Mn Mo Na Ni P Pb S Sb Se Sn U V Zn Zr C 3579±451.41 7 28±2.34 78±6.73 62606±2804.9 0 1 19±4.07 73±12.44 5363±708.59 <3.3 22705±846.49 8392±785.09 967±103.6 2±0.29 3903±226.47 15±3.76 18560±1501.8 2±0 5336±317.84 4±0.31 <9 <6.8 5±0.31 16±1.8 446±42.09 1±0.36 CNZ10 8051±306.49 <6.75 21±0.09 183±9.38 57704±1504.5 0 <0.75 31±8.47 75±7.08 4313±340.04 <3.3 23579±1027.7 5903±201.78 652±21.6 2±0.12 4254±174.98 15±1.96 13136±175.38 5±0.54 3914±133.8 <4.1 <9 <6.8 4±0.16 12±0.39 333±0.4 0±0.06 CNZ15 11080±549.22 7 19±2.58 241±17.46 45350±4727.9 0 <0.75 31±4.59 90±8.86 4560±208.14 <3.3 25412±1234.3 5482±334.69 603±26.2 2±0.11 4673±455.46 18±2.39 11421±1154.3 7±1.53 3510±346.2 <4.1 <9 <6.8 4±0.22 12±0.36 304±28.33 0±0.02 CMZ10 8803±262.76 10 21±1.81 205±2.84 55601±4824.9 0 <0.75 35±10.79 102±2.41 4569±226.44 <3.3 22591±2260.4 6506±301.57 668±26.1 2±0.16 3999±374.32 17±2.17 13675±698.8 5±0.38 4199±368.65 <4.1 <9 <6.8 5 14±1.38 343±14.99 1±0.78 CMZ15 11114±544.92 <6.75 19±0.83 242±11.08 49320±2477.3 0 <0.75 52±6.24 107±6.85 4720±258.22 <3.3 21552±128.74 6371±104.01 649±35.6 2±0.08 3895±257.65 18±3.14 12607±148.87 8±2.56 3797±237.73 <4.1 <9 <6.8 4±0.13 13±0.64 314±3.09 0±0.06 68 Table A9: Analysis of Variance (ANOVA) of shoot parameters for planting experiment Shoot parameter F-Value P-Value Biomass 142.793 0.000 Height 72.871 0.000 N 43.978 0.000 Ca 166.359 0.000 Mg 132.548 0.000 K 8.236 0.000 Fe 113.379 0.000 Cu 12.600 0.000 Mn 887.003 0.000 P 27.715 0.000 S 16.809 0.000 Zn 44.637 0.000 Table A10: Analysis of Variance (ANOVA) of root parameters for planting experiment Root parameter F-Value P-Value Root Biomass (g/kg) 114.056 0.000 Root N (mg/kg) 312.048 0.000 Root Ca (mg/kg) 31.719 0.000 Root Mg (mg/kg) 32.833 0.000 Root K (mg/kg) 70.797 0.000 Root Fe (mg/kg) 63.391 0.000 Root Cu (mg/kg) 19.487 0.000 Root Mn (mg/kg) 388.144 0.000 Root Fe (mg/kg) 63.391 0.000 Root P (mg/kg) 13.954 0.000 Root S (mg/kg) 30.365 0.000 Root Zn (mg/kg) 49.114 0.000 69